A method for the enantioseparation of radezolid (RAD), an analogue of a truly new class of antibacterial agents, oxazolidinones, was developed based on capillary electrokinetic chromatography using a cyclodextrin as a chiral pseudophase (CD-cEKC). The mechanism of RAD separation, together with its precursor, were investigated to directly define the relationship between the oxazolidinone structure and the complexation process. During the development of the method, anionic single isomer cyclodextrins were tested. They were ranked in order from hydrophilic to hydrophobic as follows: heptakis-(2,3-dihydroxy-6-sulfo)-β-cyclodextrin (HS-β-CD), heptakis-(2,3-diacetyl-6-sulfo)-β-cyclodextrin (HDAS-β-CD) and heptakis-(2,3-dimethyl-6-sulfo)-β-cyclodextrin (HDMS-β-CD). Experiments were performed at pH values of 2.5, 6.6, 8.2 and 9.6. The cyclodextrins that had an acetyl or methyl group at the C2 and C3 positions, referred to as HDAS-β-CD and HDMS-β-CD, respectively, exhibited partial and baseline separation of enantiomers in a low pH buffer. However, higher temperatures were required for HDAS-β-CD and acetonitrile addition was required for HDMS-β-CD. During the experiments, different organic solvents, varying in their amphiprotic or aprotic nature, were tested. The best results for the separation of enantiomers using the CD-cEKC method were obtained with 40 mM HDMS-β-CD dissolved in a 50 mM phosphate buffer (pH 2.5) with the addition of acetonitrile (65:35, v/v) at 27 °C, reversed polarity and a voltage equal to 28 kV. The apparent binding constants for each enantiomer to HDAS-β-CD or HDMS-β-CD were calculated. Finally, the stereochemistry of (S) and (R)-RAD and the behaviour of selected complex formations were established using electronic circular dichroism.